Method for determining platelet inhibitor response

ABSTRACT

An apparatus is provided for performing an activated clotting time test on a sample of blood containing platelets using a plunger sensor technique, the apparatus comprising a test cell, and wherein the test cell comprises an anticoagulant, a contact activator, and a predetermined amount of a platelet inactivating agent.

CROSS-REFERENCE TO OTHER APPLICATIONS

[0001] This patent application is a divisional application of U.S.patent application Ser. No. 09/225,051, filed Jan. 4, 1999, and entitled“Method for Determining Platelet Inhibitor Response,” which is acontinuation-in-part of U.S. patent application Ser. No. 08/640,277,filed Apr. 30, 1996, and entitled “Method For Determining PlateletInhibitor Response”, now abandoned, all of which are specificallyincorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates to measuring and determining theeffectiveness of antiplatelet reagents or platelet function inhibitorsin the coagulation of blood.

[0004] More specifically, the present invention relates to a method ofdetermining the effectiveness of antiplatelet reagents or plateletinhibitors on the mechanical activation of platelets.

[0005] 2. Description of the Prior Art

[0006] Blood coagulation is a complex chemical and physical reactionwhich occurs when blood comes into contact with an activating agent,such as an activating surface or an activating reagent. In accordancewith one simplified conceptual view, the whole blood coagulation processcan be generally viewed as three activities: agglutination of platelets,blood clotting, and fibrous tissue formation. In vivo, platelets flowthrough the blood vessels in an inactivated state because the bloodvessel lining, the endothelium, prevents activation of platelets. When ablood vessel is damaged, however, the endothelium loses its inertcharacter and platelets are activated by contact with tissue underlyingthe damaged site. Activation of the platelets causes them to become“sticky” and adhere together. Additional platelets then adhere to theactivated platelets and also become activated. This process continuesuntil a platelet “plug” is formed. This platelet plug then serves as amatrix upon which blood clotting proceeds.

[0007] If the chemical balance of the blood is suitable, thrombin isthen produced which causes conversion of fibrinogen to fibrin, whichforms the major portion of the clot mass. During clotting, additionalplatelets are activated and trapped in the forming clot, contributing toclot formation. As clotting proceeds, polymerization and cross-linkingof fibrin serves as the permanent clot. Thus, platelet activation playsa very important function in blood coagulation.

[0008] A number of different medical apparatuses and testing methodsexist for measuring and determining coagulation and coagulation-relatedactivities of blood. These apparatuses and methods provide valuablemedical information to an attending physician. For example, theinformation assists a physician in prescribing medication, predictingpost-operative bleeding and prescribing various therapies. Some of themore successful techniques of evaluating blood clotting and coagulationare the plunger techniques illustrated by U.S. Pat. No. 4,599,219 toCooper et al., U.S. Pat. No. 4,752,449 to Jackson et al., and U.S. Pat.No. 5,174,961 to Smith, all of which are assigned to the assignee of thepresent invention, and all of which are incorporated herein byreference.

[0009] Automated apparatuses employing the plunger technique formeasuring and detecting coagulation and coagulation-related activitiesgenerally comprise a plunger sensor cartridge or cartridges and amicroprocessor controlled apparatus into which the cartridge isinserted. The apparatus acts upon the cartridge and the blood sampleplaced therein to induce and detect the coagulation-related event. Thecartridge includes a plurality of test cells, each of which is definedby a tube-like member having an upper reaction chamber where a plungerassembly is located and where the analytical test is carried out, and areagent chamber which contains a reagent or reagents. For an activatedclotting time (ACT) test, for example, the reagents include anactivation reagent to activate coagulation of the blood. A plug memberseals the bottom of a reagent chamber. When the test commences, thecontents of the reagent chamber are forced into the reaction chamber tobe mixed with the sample of fluid, usually human blood or itscomponents. An actuator, which is a part of the apparatus, lifts theplunger assembly and lowers it, thereby reciprocating the plungerassembly through the pool of fluid in the reaction chamber. The plungerassembly descends on the actuator by the force of gravity, resisted by aproperty of the fluid in the reaction chamber, such as its viscosity.When the property of the sample changes in a predetermined manner as aresult of the onset or occurrence of a coagulation-related activity, thedescent rate of the plunger assembly therethrough is changed. Upon asufficient change in the descent rate, the coagulation-related activityis detected and indicated by the apparatus.

[0010] Certain discoveries have recently been made which contribute to abetter understanding of the role of platelets in an activated clottingtime (ACT) test. Such discoveries suggest that the activation of theplatelets has a significant and previously unappreciated effect on ACTtest results. It has long been suspected that platelet activationcontributes to total blood coagulation times, but there has been notechnique available for confirming and quantifying the impact ofplatelet activation on ACT. It has been discovered that the initialcontact and interaction of the blood sample with the activating reagenthas an impact on the platelet activation, which may make the ACT testresults variable and operator dependent. Without understanding andcontrolling the effect of platelet activation on the ACT there is aprobability of inconsistent and inaccurate clinical test results. Thedegree and extent of the impact may depend on operator technique, butthe impact and the extent of the impact has been unappreciated andunquantified. No previously known ACT test has intentionally taken intoaccount the effect of platelet activation on the test results.

[0011] In developing and testing the improved high sensitivitycoagulation detection apparatus employing the plunger technique,described in U.S. Pat. No. 5,174,961, (referred to hereinafter as the“second generation ACT apparatus”) identical blood samples were testedon both the second generation ACT apparatus and the assignee's earlierapparatus, illustrated by that described in U.S. Pat. No. 4,752,449(referred to hereinafter as the “first generation ACT apparatus”). Whenresults obtained by the second generation ACT apparatus were comparedwith the results obtained from the first generation ACT apparatus, itwas discovered that with identical blood samples and identical testcartridges, the second generation ACT apparatus produced consistentlylonger coagulation times than the first generation ACT apparatus. It wasalso noted that a still earlier and different version of the assignee'sapparatus (referred to hereinafter as the “HMS apparatus”) producedcoagulation times similar to those of the second generation ACTapparatus. The principal difference noted between the first and secondgeneration of ACT apparatus, that also differentiated the firstgeneration ACT apparatus from the HMS apparatus, was that both thesecond generation ACT apparatus and the HMS apparatus reciprocate theplunger at a relatively high rate, while the first generation ACTapparatus reciprocates the plunger at a relatively slower rate, duringinitial mixing of the sample with the activation reagent. The fasterreciprocation rate of the second generation ACT apparatus is desirableduring testing because the faster rate results in increased sensitivityfor detection of clot formation during the test.

[0012] While the reasons for the discoveries described herein may not becompletely understood at this time, it is believed that upon initialcontact of the blood with the activating reagent, the platelets are verysusceptible to the amount and type of agitation they encounter. Inaccordance with this belief, platelets are visualized as coming intocontact with activating particles and being activated at the surface ofthe particle. Slowly lowering the flag-plunger assembly leads to thegeneration of low shear forces within the blood sample bringing theplatelets into better contact with the activating particles thusallowing the platelets to adhere to the particles, achieving fullactivation. Under these conditions the platelets in the blood samplerapidly activate in which they expose platelet factor 3 activity andpossibly release platelet factor 4 activity. Under the conditions of theassay, activation of both of these components will shorten the bloodclotting time. However, if the shear rate in the blood sample is changedto higher shear forces by increasing the fall rate of the flag-plunger,the more violent agitation may strip the platelets from the surface ofthe activating reagent particles before the platelets are fullyactivated, thus prolonging or adding the previously unappreciatedvariability of some measure of uncontrolled platelet activation to theamount of time required to achieve clotting in an ACT test.

[0013] Although previous apparatuses using the plunger sensing techniquehave proven generally satisfactory, the need for certain enhancementshas been identified. Specifically, while these techniques can measureand detect coagulation and coagulation-related activities in a sample ofblood, none are designed to conveniently measure and determine theeffectiveness of antiplatelet reagents or platelet function inhibitorson the mechanical activation of platelets.

[0014] A need therefore exists for a method of determining theeffectiveness of therapeutic levels of platelet function inhibitors in avariety of circumstances. Until this invention, no such devices ormethods existed.

SUMMARY OF THE INVENTION

[0015] It is the principal object of the present invention to provide animproved method for measuring and determining the effectiveness ofantiplatelet reagents or platelet inhibitors on the coagulation ofblood.

[0016] A further object of the present invention is to provide a methodof determining the effectiveness of antiplatelet reagents or plateletfunction inhibitors on the mechanical activation of platelets.

[0017] Additional objects, advantages and novel features of thisinvention shall be set forth in part in the description that follows,and in part will become apparent to those skilled in the art uponexamination of the following specification or may be learned by thepractice of the invention. The objects and advantages of the inventionmay be realized and attained by means of the instrumentalities,combinations, and methods particularly pointed out in the appendedclaims.

[0018] To achieve the foregoing and other objects and in accordance withthe purposes of the present invention, as embodied and broadly describedtherein, the present invention is directed to a method of determining adose response for a platelet inhibitor. The method includes the steps ofplacing a predetermined amount of heparin in each cell of a multicelltest cartridge, placing an optimized amount of a mechanical plateletand/or clotting activator in each cell, and placing a measured amount ofplatelet inhibitor in each cell, the amount of inhibitor in each celldiffering from the amount in each other cell. An aliquot of a bloodsample is added to each cell, and the blood sample aliquot, plateletand/or clotting activator and platelet inhibitor are mixed. Each cellsample is allowed to clot, and the clotting time for each cell ismeasured. The relative clotting times are used to calculate anddetermine the platelet inhibition effect of the platelet inhibitor.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The accompanying drawings, which are incorporated in and form apart of the specification, illustrate the preferred embodiments of thepresent invention, and together with the descriptions serve to explainthe principles of the invention.

[0020] In the Drawings:

[0021]FIG. 1 is a hypothetical graph of Percent Inhibition vs. InhibitorConcentration.

[0022]FIG. 2 is a perspective view of a 6-channel plunger sensorcartridge, a 4-channel plunger sensor cartridge, and a high sensitivitycoagulation detection apparatus with which the cartridges are used onselectively alternate basis, all of which comprises an apparatus formeasuring and detecting coagulation and coagulation-related factors influids, in accordance with the present invention;

[0023]FIG. 3 is a front elevational view of the 6-channel plunger sensorcartridge shown in FIG. 2; and

[0024]FIG. 4 is an enlarged vertical sectional view through one of thetest cells of the plunger sensor cartridge, taken in the plane of line4-4 shown in FIG. 3.

DESCRIPTION OF THE PREFERRED EMBODIMENT

[0025] In accordance with the present invention, it has been discoveredthat the ability of platelet inhibitors or antiplatelet drugs to effectcoagulation of blood can be readily assessed. To this end, by usingdifferent concentrations of a platelet inhibitor in a plurality of testcells, and using an optimized amount of a mechanical contact activatorof platelets and/or clotting, the ability of an inhibitor in a selecteddose to prevent the mechanical activation of platelets can be assessed.

[0026] To practice the method for measuring and determining theeffectiveness of antiplatelet reagents or platelet function inhibitorson the mechanical activation of platelets of the present invention, eachof test cells 66 (shown in FIGS. 3 and 4 and discussed in further detailbelow) includes a novel reagent composition. This novel reagentcomposition (shown in FIG. 4 as reagent composition 80 in reactionchamber 94) may be either a liquid or a solid powder, and includes twocomponents: an anticoagulant agent, and a platelet inhibitor agent.

[0027] The anticoagulant agent of the reagent composition 80 accentuatesthe rate-limiting step in coagulation. The anticoagulant agent may beany substance having anticoagulant activity and which is not affected byfactors derived (released) from activated platelet. A wide variety ofsuitable anticoagulant compounds are known and are readily available inthe art including, for example, but not limited to, heparin, argatrobanand other synthetic or natural compounds which are specific inhibitorsof thrombin and/or clotting factor Xa. Preferably, the anticoagulantagent is a substrate-derived competitive thrombin inhibitor, such assynthetic peptides, arginine derivatives, benzamidine derivatives, andlysine derivatives. In a particularly preferred embodiment, heparin isthe anticoagulant agent of choice since it is necessary to inhibitreactions which occur later in the coagulation process, such asactivation of factors X, II (prothrombin) and I (fibrinogen), i.e.,reactions in the common pathway or in a later stage in the intrinsicpathway. By inhibiting these later clotting factors, heparin (whichtogether with antithrombin III accelerates the inactivation of factor Xand thrombin) helps to accentuate the contribution of platelets toclotting, which is the rate-limiting step in blood coagulation, i.e.,the actual clotting times in the ACT test depend on how rapidly theplatelets are able to activate clotting. Accordingly, heparin must beeither present in the blood sample or included in the reagentcomposition 80 to inhibit non-platelet-related reactions which tend toobscure the rate-limiting activation step.

[0028] The concentration of the anticoagulant agent in reagentcomposition 80 required to achieve sufficient test sensitivity willdepend upon the anticoagulant activity of the particular agent and thelevel of sensitivity desired. With the presently preferred anticoagulantagent, heparin, the reagent concentration is preferably between about 1to 4 units/ml, and more preferably between about 2 to about 4 units/ml.

[0029] The final component of the novel reagent composition 80 of thepresent invention is a platelet inhibiting agent which serves as thechemical platelet inhibitor to facilitate evaluation of the function ofplatelets in the blood sample to be tested. If the platelets areinactive or not functioning normally, the platelet inhibiting agent willhave a lessened or no effect on the clotting time. Platelet inhibitingagents are well known and readily available in the art. Two classes ofplatelet inhibitors exist; the first class comprises compounds that acton platelet membrane sites, broadly known as IIa/IIIb inhibitors suchas, but not limited to, Abciximab, which is the Fab fragment of thechimeric human-murine monoclonal antibody 7E3 and sold under thetrademark ReoPro™, or4-[4-[4-(aminoiminomethyl)phenyl]-1-piperazinyl]-1-piperidineaceticacid, hydrochloride trihydrate sold under the trademark GR144053™. Thesecond class comprises compounds that are metabolic inhibitors such as,but not limited to, acetylsalicylic acid or aspirin.

[0030] The specific concentrations of the platelet inhibiting agent inreagent composition 80 of the present invention (i.e., reagentcomposition 80 in reaction chamber 94 of the test cell 66, shown in FIG.4) will depend, in part, upon the specific antiplatelet compound to betested, but regardless varying amounts will be used in each test cells66 so that selected doses can be assessed for the ability to preventmechanical activation of platelets.

[0031] As discussed previously, reagent composition 80 of the presentinvention may be either a liquid or a solid powder, although a solidpowder form (commonly referred to as a “dry fill”) is preferred. Toprepare reagent composition 80 as a solid or dry fill, predeterminedamounts of the two components (anticoagulant agent, and plateletinactivating agent) are combined and mixed with a saline (NaCl) solutionpreferably containing a protein, such as bovine serum albumin, tostabilize the platelet inactivating agent. The mixture may be diluted,if necessary, with additional saline solution to achieve the desiredplatelet inactivating agent concentrations. A predetermined amount ofeach solution of reagent composition 80 is placed in the reactionchambers 94 of the test cells 66 (shown in FIGS. 2, 3 and 4) and allowedto evaporate, leaving a solid or dry fill residue of reagent composition80.

[0032] The method and apparatus of the present invention also includes acontact activator in the reagent chamber (shown in FIG. 4 as reagentchamber 92) of each test cell 66. The contact activator (discussed belowand shown in FIG. 4 as contact activator 90) includes an activator(commonly referred to as a surface activator), such as kaolin, toactivate platelets and blood Factors XII and/or XI. However, as will beappreciated by those of skill in the art, other contact activators whichfunction in a similar manner to kaolin may be used for the practice ofthe invention, such as diatomaceous earth, powdered glass, silica, orany other particle having a negatively charged surface. Activators maybe chosen by simply performing the method of the present invention withdiffering activators and comparing the magnitude of clotting time.Kaolin is preferable however, since it is an activator of bothcoagulation and platelets. If desired, one could also use a contactactivator of platelets in combination with a contact activator ofcoagulation. The contact activator 90 may be present in either a solidor liquid form, although the presently preferred reagent is in a liquidform. To prepare a liquid contact activator 90, the activator componentmay be dissolved in an appropriate buffered solution, a variety of whichare known to those skilled in the art, including HEPES(hydroxyethyl-piperazine ethanesulfonic acid) buffer. A bacteriostaticagent such as sodium azide may also be included in clotting reagent 90.In a particularly preferred embodiment, contact activator 90 compriseskaolin, HEPES buffer, calcium chloride (to achieve a linear clottingtime response to heparin), and sodium azide as the bacteriostatic agent.Kaolin in the amount of 2% to 15% and preferably 4% to 12% may be used.One illustrative composition is HEPES buffer, 50 mM calcium chloride,0.02% sodium azide as a bacterio-static agent, and 4% kaolin, at pH 7.4.

[0033] In order to provide a series of differing clotting times, atleast two of the test cells comprise different amounts of the plateletinactivating agent. In the exemplified embodiment shown in FIG. 3, thefirst two cells 66A and 66B (which represent the “baseline” ornon-activated clotting time) contain no platelet inhibiting agent. Eachsuccessive cell 66C, 66D, 66E, and 66F includes increasing amounts ofplatelet inhibiting agent. Although the concentrations and proportionsmay vary depending upon the particular reagent components (as discussedabove), the following table provides an illustration of suitable amountsof the presently preferred components in reagent composition 80, priorto dilution with the blood sample: TABLE 1 Amounts of Components inReagent Composition 80 (Test Cartridge 64) Cell Cell Cell Cell Cell CellReagent 66A 66B 66C 66D 66E 66F Platelet 0.0 ng 0.0 ng X aX bX cXInhibitor Heparin 3.0 U 3.0 U 3.0 U 3.0 U 3.0 U 3.0 U

[0034] The actual concentrations cannot be given until a specificantiplatelet compound is tested in the system. X represents a lowconcentration of the compound with which inhibition (or an increase inthe clotting time) is just noticeable. Factors a, b, and c representmultiple increases in the amount of the compound X added to the cells.Concentration cX is preferably an excess of the compound, in order togive maximal inhibition of platelet activation. Increasing the amount ofthe antiplatelet drug above this concentration will not further increasethe clotting time.

[0035] To determine the platelet inhibition in accordance with oneembodiment of the present invention, a predetermined volume of blood tobe analyzed is introduced into the reaction chamber 94 of each test cell66A-66F, thereby dissolving reagent composition 80. The contactactivator 90 in each reagent chamber 92 is then introduced into thecorresponding reaction chamber of each test cell 66A-66F, at a low shearforce mode. At the termination of the “mix cycle,” the agitator isswitched to a drop rate in the high shear force mode and the clottingtime is determined. The clot ratio is then calculated based on theclotting time for each test cell. When all six cells have clotted, thetest is terminated and a relative clotting time is computed wherein theclotting times of the cells containing no platelet inhibitor are used asthe reference clotting time. The simplest calculation is to considerthis zero percent inhibition, and the cell with the maximum amount orexcess of the inhibitor is considered as 100% inhibition. The clottingtimes from the intermediate cells are then compared to determine thedose response.

[0036] Referring now to FIGS. 2, 3 and 4, the presently preferredembodiment of an apparatus 62 and a plunger sensor cartridge 64 may beused together in order to evaluate the effectiveness of antiplateletreagents or platelet inhibitors on the mechanical activation ofplatelets. The apparatus 62 and an alternate cartridge 65 can also beused to perform a platelet inhibition test using these unique reagents.In general, the ACT test and other heparin-protamine titration testswhich may be performed using the apparatus 62 and the cartridges 64, 65have previously been described in the assignee's aforementioned patentsand applications, including U.S. Pat. No. 4,599,219 and U.S. Ser. No.08/640,275, now issued as U.S. Pat. No. 5,925,319, the disclosures ofwhich are incorporated herein by reference. Accordingly, many of thedetails of functionality will be generalized herein with theunderstanding that the assignee's prior patents and applicationsdisclose many of these details to a greater extent. It is anticipatedthat similar results and effects as those obtained from using theassignee's plunger sensor technique will also be obtainable bypracticing the present invention using other well known methods anddevices.

[0037] In general, the cartridges 64, 65 as shown in FIGS. 2 and 3include a plurality of test cells 66 (shown in FIG. 4), each of which isformed generally as a downward extending truncated tube-like member 68.Each of the tube-like members 68 is connected to an upper shelf portion70. A plunger assembly 72 (shown in FIG. 4) extends downward from anupper open end of each test cell 66 into the tube-like member 68. Eachplunger assembly 72 includes at least one and preferably a pair of flags74 at the upper end located at a position above the shelf portion 70.The plunger assembly 72 also includes a shaft 76 which extends from theflags 74 downward to a lower end upon which a disk member 78 isattached. The disk member 78 is formed of resilient material andincludes a center, generally cylindrical main body portion 82 and anannular flange 84 located above and extending outward from the main bodyportion 82. The annular flange 84 includes slots or openings (not shown)formed therein at outer circumferential locations.

[0038] As shown in FIG. 4, prior to using the plunger sensor cartridge64 or 65 in the apparatus 62, the disk member 78 is positioned with itsmain body portion 82 located in and sealed against an opening formed bya partition 86 extending inwardly from the tube-like member 68. Thepartition 86 is located between the upper and lower open ends of thetube-like member 68. A resilient flexible plug 88 is positioned in theinterior of the tube-like member at its lower open end. The plug 88seals against the inner side walls of the tube-like member 68 andconfines a quantity of contact activator 90 (discussed above) in areagent chamber 92 between the partition 86 and the plug 88. Theclotting reagent 90 may be a liquid or a solid powder. A reactionchamber 94 is generally defined by that portion of the open tube-likemember 68 above the partition 86. A predetermined amount of a reagentcomposition 80, the contents of which have been described in detailabove, is provided in the reaction chamber 94 as either a liquid or asolid powder.

[0039] The plunger sensor cartridge 64 or 65 is inserted into areceiving block (not shown) in the apparatus 62 to conduct the ACT test.Each of the test cells 66 extends into a receptacle 98 of the receivingblock. Each receptacle 98 has a configuration adapted to receive a testcell 66, while the shelf portion 70 of the cartridge 64 or 65 sits ontop of the receiving block.

[0040] The apparatus 62 is generally formed of subassemblies. Adispensing subassembly 104 of the apparatus 62 automatically supplies asample of blood to each test cell 66 of the cartridge 64 or 65. Thereagent composition 80 is dissolved in the blood sample when the bloodsample is introduced into the reaction chamber 94. A plunger liftingassembly 99 of the apparatus 62 controls the lifting movement of theplunger assembly 72, and a reagent drive subassembly 100 of theapparatus 62 moves the plug 88 to force the clotting reagent 90 into thereaction chamber 94, thereby mixing the clotting reagent 90 with thereagent composition 80. An optical sensing system (not shown) senses thephysical descent of the plunger assembly 72 through the blood sample andreagent mixture in the reaction chamber 94 in order to detectcoagulation condition.

[0041] The sample of blood is supplied to the reaction chamber 94 from asyringe 102 having a blunt needle 103 attached thereto. The syringe 102is manually attached to the dispensing subassembly 104 of the apparatus62. The body of the syringe 102 contains blood, preferably fresh drawnfrom the patient, upon which the ACT test is to be performed. Of course,prior to attachment of the syringe 102 to the dispensing subassembly104, all air or other voids in the blood within the syringe 102 and theblunt needle 103 is removed in the conventional manner. A plunger 106located within the body of the syringe 102 is engaged with a drive wheel108. Rotation of the drive wheel 108 forces the syringe plunger 106downward and expels a predetermined amount of blood from the lower endof the blunt needle 103. The extent to which the syringe plunger 106 ismoved downward determines the quantity of blood expelled from theneedle.

[0042] The dispensing subassembly 104 includes a movement frame 110which is moved laterally in the horizontal direction along guide rods112. The degree of lateral movement is controlled by the microprocessor(not shown) of the apparatus 62 in accordance with programmedinformation, thereby locating the blunt needle 103 directly above theopen upward ends of each test cell 66 of the cartridge 64 or 65. Afterattaining the proper lateral location, the movement frame 110 moves thesyringe 102 vertically downward to insert the lower end of the bluntneedle 103 into each of the test cells 66. The desired amount of fluidsample is automatically dispensed into the test cell 66. Thereafter, theblunt needle 103 is withdrawn from the test cell 66 by the movementframe 110, and the next lateral position over a test cell 66 is assumed.The sequence again repeats itself, thereby injecting into each test cell66 of the plunger sensor cartridge 64 or 65 that predetermined amount ofblood sample needed for conducting the ACT-type test.

[0043] A portion of the plunger lifting subassembly 99 is shown in FIG.2, and includes at least one and preferably a plurality of lift wires114. The lift wires 114 are positioned in a lowermost location, and inthat position a horizontal segment of the lift wires 114 fits underneaththe flags 74 of the plunger assembly 72. Upward movement of the liftwires 114 lifts each of the plunger assemblies 72 upward, therebyremoving the disk member 78 from its sealed location in the opening 84of the partition 86. A fluid communication passageway through theopening 84 between the reagent chamber 92 and the reaction chamber 94 isthereby established. The reagent drive subassembly 100 includes aplurality of plug driver shafts 116 (shown in FIG. 4). Thereafter, orsimultaneously with the upward movement of the plunger assembly 72, theplug driver shafts 116 of the reagent drive subassembly 100 move upward,forcing each plug 88 upward collapsing the reagent chamber 92 andforcing its contents 90 into the reaction chamber 94.

[0044] At the commencement of the ACT-type test, a sample of blood uponwhich the test is to be performed is introduced into the reactionchamber 94. The lift wires 114 of the subassembly 99 lift the plungerassembly 72 to withdraw the disk member 78 from its seated engagementwith the opening in the partition 86. The plug 88 is pushed upwardagainst the partition 86 by the plug driver shafts 116 of thesubassembly 100. The contact activator 90 from the reagent chamber 92 isforced through the opening into the reaction chamber 94. The contactactivator 90 is mixed with the blood and reagent mixture in the reactionchamber 94, by reciprocating the plunger assembly 72 at a low shearforce for a predetermined period of time of about 8 to 60 seconds. Thelift wires 114 of the subassembly 99 continue to lift the plungerassembly 72, which descends by the force of gravity through the pool offluid in the reaction chamber 94. At the termination of this firstperiod of time, the plunger assembly is switched to a drop rate in ahigh shear force mode. This optimizes the ability to detect clotformation. Clotting time is then measured in each test cell. As theplunger assembly 72 descends through the fluid it is resisted by aproperty of the fluid in the reaction chamber 94, such as the viscosity,which changes as a result of the onset or occurrence of acoagulation-related activity, e.g., platelet activation and aggregationfollowed by coagulation resulting in fibrin formation. The descent rateof the plunger assembly 72 therethrough is changed, which indicates theoccurrence of a coagulation-related activity.

[0045] When all six cells have clotted, the test is terminated and arelative clotting time is computed wherein the clotting times of thecells containing no platelet inhibitor are used as the referenceclotting time. The simplest calculation is to consider this zero percentinhibition, and the cell with the maximum amount or excess of theinhibitor is considered as 100% inhibition. The clotting times from theintermediate cells are then compared to determine the dose response.

[0046] The purpose of the foregoing test is three-fold: 1) to determinean individual's basic response to the platelet inhibitor, 2) to monitorthe presence of the platelet inhibitor, and 3) to determine thequantitative concentration of the inhibitor.

[0047] To illustrate the use of the present invention, a dose responsecartridge test is performed on a patient using compound X as a specificplatelet inhibitor. A titration curve is plotted which is used tocompute a dosage of the drug which will be given to the patient. Thetest apparatus computes this dosage based on the clotting times and theprogram which relates to this specific drug. After giving the patientthe drug, a two channel activated clotting time assay is performed. Thismeasures whether or not the target clotting time has been achieved andis a measure of the effectiveness of the drug.

[0048] If a specific concentration of the drug is desired, this may bedetermined by using the previous dose response curve obtained to computethe concentration of the inhibitor. Alternatively, if a chemical orantibody exists which will neutralize the platelet inhibitor, theconcentration of the platelet inhibitor can be determined using atitration cartridge containing varying amounts of the neutralizingchemical.

[0049]FIG. 1 illustrates a process in which the desired degree ofplatelet inhibition is a 50% inhibition. The Y-axis is computed from theclotting times obtained using the above-described dose response assaycartridge. The clinician enters the 50% inhibition figure into theapparatus and the apparatus computes the information given in FIG. 1.Using FIG. 1, the horizontal line labeled “Desired Inhibition Level”represents 50% inhibition. This line intersects the “Dose ResponseCurve.” A vertical line is dropped from this point of intersection tothe X-axis where the intersection of the line with the X-axis providesthe concentration of inhibitor required to achieve 50% inhibition of theplatelets. FIG. 1 is labeled in terms of cell numbers, but could also belabeled with concentrations of the inhibitor, from zero in cell 1 to themaximum (100% inhibition) in cell 6.

[0050] In monitoring the effectiveness of the drug, the above doseresponse curve can be used after running a two channel assay cartridge,taking the clotting time (the Y-axis could also be given in terms of theclotting times rather than a percentage of inhibition). This is used toconvert the clotting time to a concentration of the inhibitor using thesame type of calculation as above.

[0051] A third method for determining the amount of platelet drugpresent uses a titration assay. This requires that a chemical orantibody capable of neutralizing the inhibitor be available. In atitration cartridge, each channel of the assay cartridge containsincreasing amounts of the neutralizing agent. The channel where theshortest clotting time is obtained is the concentration of neutralizingagent where all of the platelet inhibitor has been neutralized. Knowingthe stoichiometry between the neutralizing agent and the plateletinhibitor, the concentration of the platelet inhibitor present in thesample can be computed.

[0052] The foregoing description is considered as illustrative only ofthe principles of the invention. Furthermore, since numerousmodifications and changes will readily occur to those skilled in theart, it is not desired to limit the invention to the exact constructionand processes shown as described above. Accordingly, all suitablemodifications and equivalents may be resorted to falling within thescope of the invention as defined by the claims which follow.

The embodiments of the invention in which an exclusive property orprivilege is claimed are defined as follows:
 1. An apparatus forperforming an activated clotting time test on a sample of bloodcontaining platelets using a plunger sensor technique, said apparatuscomprising a test cell, and wherein said test cell comprises ananticoagulant, a contact activator, and a predetermined amount of aplatelet inactivating agent.
 2. The apparatus of claim 1 , wherein theanticoagulant is heparin.
 3. The apparatus of claim 2 , wherein theamount of heparin in said cell is between about 1 unit and about 4 unitsper milliliter of blood sample.
 4. The apparatus of claim 1 , whereinthe contact activator is a particle having a negatively charged surface.5. The apparatus of claim 3 , wherein said particle is kaolin.
 6. Theapparatus of claim 4 , wherein the amount of contact activator in saidcell is between about 2 percent and about 15 percent.
 7. The apparatusof claim 4 , wherein said particle is diatomaceous earth.
 8. Theapparatus of claim 4 , wherein said particle is powdered glass.
 9. Theapparatus of claim 4 , wherein said particle is silica.
 10. Theapparatus of claim 1 , wherein said platelet inactivating agent isselected from the group consisting of Abciximab,4-[4-[4-(aminoiminomethyl)phenyl]-1-piperazinyl]-1-piperidineaceticacid, hydrochloride trihydrate, and acetylsalicylic acid.
 11. Anapparatus for performing an activated clotting time test on a sample ofblood, said apparatus comprising a plurality of test cells, said cellsbeing adapted for receiving an aliquot portion of said sample, whereineach of said cells comprises an anticoagulant and a plateletinactivating agent, and wherein at least one of said cells furthercomprises a clotting activator, wherein a clotting time is determinedfor each of said aliquot portions, and wherein a relative clotting timefor each of said aliquot portions comprising the clotting activator isdetermined as compared to a reference clotting time for said at leastone cell containing no clotting activator, wherein said relativeclotting times in said cells are determinative of the clottingactivation of said clotting activator.
 12. The apparatus of claim 11 ,wherein the anticoagulant is heparin.
 13. The apparatus of claim 12 ,wherein the amount of heparin in said cell is between about 1 unit andabout 4 units per milliliter of blood sample.
 14. The apparatus of claim11 , wherein the contact activator is a particle having a negativelycharged surface.
 15. The apparatus of claim 13 , wherein said particleis kaolin.
 16. The apparatus of claim 14 , wherein the amount of contactactivator in said cell is between about 2 percent and about 15 percent.17. The apparatus of claim 14 , wherein said particle is diatomaceousearth.
 18. The apparatus of claim 14 , wherein said particle is powderedglass.
 19. The apparatus of claim 14 , wherein said particle is silica.20. The apparatus of claim 11 , wherein said platelet inactivating agentis selected from the group consisting of Abciximab,4-[4-[4-(aminoiminomethyl)phenyl]-1-piperazinyl]-1-piperidineaceticacid, hydrochloride trihydrate, and acetylsalicylic acid.